We have recently reported the development of an ultrasound-assisted gene transfer (UAGT) method in the salivary gland. This technique enables transient, non-invasive, non-viral gene transfer to the salivary gland using off the shelf FDA-approved microbubbles and clinical ultrasonography equipment. We submit that this technology may prove enabling for applications in gene therapy for Xerostomia given the limitations of viral vector systems, which include sialoadenitis and consequent hyposalivation. Considering the substantial prior work that has been performed in miniature swine as a close-to- man model of salivary gland function, our first aim will be to upscale our UAGT technique in miniature swine and index resultant gene expression to viral vectors. The overall goal of this first aim will be to show equivalence to viral vectors, thereby providing a rationale for their replacement. We will also test in our system an advanced-generation (minicircle) plasmid with the putative capability of longer-lasting gene expression following non-viral gene transfer. Our next step will be to undertake a restorative gene therapy treatment in our swine subjects utilizing Aquaporin-1 (AQP1), the therapeutic transgene that has shown salivary restorative promise in an ongoing human gene therapy clinical trial. UAGT and viral methods will be utilized in parallel, with primary experimental metrics being functional, namely salivary flow. Animals will be irradiated according to previously published methodologies. AQP1 treatment with viral vectors (Adenovirus and Adeno- associated virus) will be undertaken as previously described and will serve as an index by which to measure the efficacy of our UAGT-based AQP1 gene therapy. In contrast to earlier studies, we will endeavor retreatment when salivary flow declines to <20% of that in the contralateral (control) gland, with our goal being maintenance of functional improvement for 12 months. We will thereby test our hypothesis that UAGT will enable chronic maintenance of restorative gene therapy through retreatment, whereas viral vectors will not. In our final aim, we will explore an important but heretofore unaddressed issue, that of the proteomic quality of saliva produced by AQP1 gene therapy. To do this, we will undertake full- proteome profiling of the saliva produced in our animal subjects with AQP1 gene therapy, compared with saliva from non-irradiated swine and irradiated swine wherein partial sparing of the salivary gland has occurred. Scans will be performed using 2-dimensional difference gel electrophoresis, supplemented by multi- dimensional chromatography and mass spectrometry methods. PUBLIC HEALTH RELEVANCE: This application will implement a gene therapy strategy for radiation-induced xerostomia, or dry mouth, a nearly inevitable consequence of head and neck radiation affecting thousands of cancer survivors. The first human clinical trial involving salivar gland gene therapy is now underway, utilizing an Adenoviral vector expressing the water channel Aquaporin-1 as a means of increasing salivary flow. Our application is designed to improve upon this strategy by utilizing a novel ultrasound-assisted gene transfer method to deliver the Aquaporin-1 gene drug, thereby obviating the toxicity of viral vectors.